Investigating Chromatin-Driven Spindle Assembly in the Drosophila melanogaster syncytial Embryo

Abstract

Faithful segregation of chromosomes between daughter nuclei is crucial for the proliferation of all eukaryotic cells. The process is facilitated by the mitotic spindle, a bipolar array ofmicrotubules (MTs) and associated proteins which exert force on chromosomes to organiseand separate them. It was originally understood that the spindle was nucleated from two centrosomes at opposing poles; however, research has elucidated multiple mechanisms of spindle assembly, active even during centrosomal spindle assembly. Amongst these, chromatin-driven spindle assembly is particularly important in centrosomal tissues and in polycentrosomal cancer cells. To date, chromatin-driven spindle assembly has been observed to require the signalling protein Ran, and a number of spindle assembly factors, including the Augmin complex, HURP, and TPX2. In Drosophila, which lacks a clear homolog of TPX2, HURP is particularly important for this process. This study used cold-induced MT depolymerisation of Drosophila embryos to elucidate the involvement of key microtubule-associated proteins in this pathway of spindle assembly. Members of the γ-Tubulin ring complex were observed to localise to MTs nucleated proximal to chromatin, indicating the likely requirement for the complex for initial MT nucleation during chromatin-driven spindle assembly. Injection of RNA for dHURP truncations prior to observation of this pathway displayed that the protein does not localise to chromatin as previously reported, instead localising to the spindle envelope, which coalesces around chromatin upon MT depolymerisation. This observation, in conjunction with immunoprecipitation followed by tandem mass spectrometry (IP-LC-MS/MS) of dHURP truncations to elucidate putative interactors, has inspired a hypothesis for recruitment of SAFs to the vicinity of chromatin via the spindle envelope/matrix its and associated proteins. Finally, investigation of the localisations of processive motors indicated their involvement in this pathway of spindle assembly, and IP-LC-MS/MS elucidated their potential interactors. This thesis provides valuable insight into the dynamics of key microtubule-associated proteins in chromatin-driven spindle assembly.